The ability to deliver genes using cell surface receptors to achieve organ specific targeting in vivo has been demonstrated, although with low efficiency. High level expression with DNA complexes in vitro requires adenovirus for endosome lysis and perhaps for other unidentified functions. The objective of this project is to develop chemically defined delivery systems to achieve highly efficient delivery of genes into the nucleus of the airway epithelium, first in vitro and then in vivo. By the incorporation of specific peptides and proteins in the DNA complex, this type of delivery system will facilitate an understanding of the role of viral and cellular proteins in achieving high level expression of exogenous DNA. Specific Aim 1 is to prepare DNA binding poly-L-lysine conjugates containing receptor ligands for the (a) folate receptor, (b) mannose-6- phosphate receptor (c) vasoactive intestinal peptide and (d) dimeric immunoglobulin A receptor of airway epithelial cells. Physical and chemical methods will be used to characterize the structural, thermodynamic, and kinetic properties of the DNA complexes. Specific Aim 2 is to elucidate the structural requirements for endosome lysis by lytic peptide sequences taken viral fusogenic proteins. Biochemical methods and digital imaging fluorescence microscopy will be used to determine if the peptides must form trimers to achieve pH dependent lysis of liposomes and endosomes in living cells. Specific Aim 3 is to determine, after endocytosis of the DNA:ligand complex and its release into the cytoplasm, what kind of structural and compositional changes in the complex are necessary for the DNA to move through the cytoplasm into the nucleus. DNA movement in the cytoplasm, DNA accumulation in the nucleus, and the probable dissociation of the complex in the cytoplasm will be measured using biochemical methods and digital imaging fluorescence microscopy. Specific Aim 4 is to synthesize DNA binding ligands that facilitate transport of the DNA through the cytoplasm into the nucleus. The SV-40 nuclear localization sequence on a DNA binding polypeptide will be tested as a component of the DNA complex. Specific Aim 5 is to demonstrate that the DNA complexes, constructed on the basis of in vitro experiments, are effective for gene delivery in vivo.